Protein sequencing has assumed a pivotal role in biological research. Partial amino acid sequence data are used to help identify and clone unknown proteins. Frequently, sequence data are used to identify the proteins that are separated by 1- or 2D gel electrophoresis, and to characterize individual components in HPLC peptide maps. Almost all high sensitivity protein and peptide sequencing is done by automated Edman degradation, where current technology limits the amount of sequence data that can be generated to less than 45 amino acid residues per day. In practice, many biological research problems require the sequence analysis of greater numbers of peptides or proteins than can be handled conveniently by conventional Edman sequencing. Thus, there is a great need for rapid protein sequencing methods. We have developed a new principle in protein sequencing that involves two steps. First, a sequence-defining set of fragments is generated in a controlled fashion. Second, matrix assisted laser desorption mass spectrometric is used to read out the complete data set in a single operation, as a protein sequencing ladder. The amino acid sequence of the parent peptide chain can be deduced from the mass difference between consecutive peptide fragments. It can be anticipated that sensitivity of the protein ladder sequencing method will be comparable to existing Edman methods. Readout of the protein ladder has been demonstrated at less than 0.1 picomoles per component and automated Edman degradation has been demonstrated on single picomole amounts of sample. Thus, extensive sequence data will be obtainable by protein ladder sequencing from low picomole total amounts of peptide or protein sample. In principle, the ladder generating chemistry can be carried out in parallel on large numbers of samples, and the laser desorption mass spectrometric read out is typically ~1 minute per sample. Conservatively, this would give a total throughput of 10-20+ residues per minute (>100 residues per hour), at very low per cycle cost. Such rapid, inexpensive sequencing could vastly expand the applications and use of protein sequence analysis in biological research. Paper describing these results has been published in SCIENCE and elsewhere.